Bacterial small RNAs (here referred to as sRNAs) are important regulators for gene expression, especially those associated with stress responses and virulence. sRNAs function by base pairing with their target mRNAs and affecting their translation and stability. The ability of each sRNA to regulate multiple targets in its regulon creates a complex network for shaping gene expression and phenotype, which ultimately leads to global adaptation of bacteria. How do the kinetic properties of sRNA-mediated regulation establish an ordered pattern of gene expression? What is the global impact of sRNA-mediated regulation on bacterial phenotypes? These are two important fundamental questions yet to be addressed. In this proposal, we will tackle these questions. One of the bottlenecks so far that has precluded the building a complete model to describe sRNA regulatory networks is the lack of kinetic measurements inside bacterial cells. Therefore, we first propose to develop a super-resolution imaging and analysis platform allowing direct visualization and characterization of sRNA and target mRNAs at the single-cell level with single copy sensitivity. Applying this imaging and analysis platform to a model sRNA system, SgrS, in E. coli, we will fully dissect kinetic mechanisms of sRNA regulation on individual targets in Aim1, and further explore the molecular mechanism that governs the regulation selectivity among multiple targets in the regulon in Aim 2. In order to understand the global impact of sRNA regulation on the bacterial phenotype, in Aim 3, we will integrate high-throughput transcriptomic data and single cell imaging data with genome scale flux balance models of E. coli metabolism and identify differential metabolic pathway usage as a result of sRNA regulation. The proposed study by the combination of multi-level experimental characterization and computational simulation will provide the most systematic description of sRNA regulation to date and will establish a novel framework for sRNA analysis that can be generalized to other bacterial and eukaryotic sRNAs.